The invention relates to the field of novel antibiotic peptides, including naturally occurring peptides. The nucleic acid sequence encoding the peptide and the corresponding amino acid sequence are included, together with methods of using the same.
Bacterial infections remain among the most common and deadly causes of human disease. Unfortunately, the overuse of antibiotics has led to antibiotic resistant pathogenic strains of bacteria. Indeed, bacterial resistance to the new chemical analogs of these drugs appears to be out-pacing the development of such analogs. For example, life-threatening strains of three species of bacteria (Enterococcus faecalis, Mycobacterium tuberculosis, and Pseudomonas aeruginosa) have evolved to be resistant against all known antibiotics. [Stuart B. Levy, xe2x80x9cThe Challenge of Antibiotic Resistancexe2x80x9d, in Scientific American, pgs. 46-53 (March 1998)]
Classical penicillin-type antibiotics bind to cell wall synthetic enzymes and thereby deregulate the activity of a single class of proteins known as autolysins which leads to bacterial lysis and bacterial cell death. The development of new drugs which affect an alternative bacterial target protein would be desirable. Pneumococcus is a particularly relevant organism for such study because 1) it has only one predominant autolysin (LytA rather than the multiple autolysins of other bacteria), 2) the autolysin has been cloned and sequenced and can therefore be easily manipulated genetically, and 3) pneumococcus has only one growth zone so that is possible to study activation of the enzyme in a fairly defined region of the cell.
Most bacteria are stabilized by a cell wall consisting of a glycopeptide polymeric murein (peptidoglycan) that completely encloses the cell [Weidel and Pelzer et al., Enzymol., 26:193-232 (1964)]. Expansion of the cell wall during bacterial growth and splitting of the septum for cell separation requires enzymes that can cleave this covalently closed network. In addition to acting as spacemaker enzymes for cell wall growth [Tomasz et al., Walter de Gruyter, 155-172 (1983)], certain murein hydrolases also act as autolysins, putative suicide enzymes. The life and death dichotomy of autolysin function demonstrates the need for efficient and strict regulation of murein hydrolase activity, a paradigm conceptually similar to that for caspases in the process of eukaryotic apoptosis. Not surprisingly, the regulation of the autolysins is a highly sophisticated physiological task. For example, the enzymes must be controlled at their extracytoplasmic location. In addition, most bacteria possess multiple hydrolases which must be controlled in concert. Antibiotics such as penicillin induce bacteriolysis by interfering with the control of the endogenous autolytic enzymes, indicating the significant chemotherapeutic relevance of these enzymes.
Although the binding of antibiotics to cell wall synthetic enzymes has been very well characterized, it is unknown how this event leads to deregulation of autolytic enzymes. During normal cell growth, autolysin activity is believed to be subject to strong, prolonged downregulation. Thus, the expression of most hydrolases is constitutive throughout the cell cycle but the enzymes are only physiologically active during stationary phase lysis [Hakenbeck and Messer, Eur. J. Biochem, 129:1239-1244 (1977); Ronda et al., Eur. J. Biochem., 164:621-624 (1987)]. Autolysin activity during exponential phase also remains curtailed even when the gene is constitutively expressed from a plasmid [Ronda et al., Eur. J. Biochem., 164:621-624 (1987)]. This indicates regulation of autolysin activity is independent of transcription of the autolysin itself. In addition, cell wall hydrolases are continuously present on the cell surface and, since triggering of wall hydrolysis does not require the synthesis of new enzyme [Kitano and Tomasz, Antimicrob. Agents Chemother., 16:838-848 (1979)], these enzymes must be prevented from potential hydrolytic activity.
The most striking example of physiological down regulation of autolysis is the stringent response that occurs during deprivation of an essential nutrient [Cashel et al., The Stringent Response, In Escherichia coli and Salmonella: Cellular Molecular Biology, Neidhardt et al., Eds., (Washington, D.C.: ASM Press, (1996)]. Starved bacteria bind antibiotic normally, but do not lyse or die. Upon starvation, bacteria rapidly accumulate guanosine 3xe2x80x2,5xe2x80x2-bispyrophosphate (ppGpp), which is synthesized by ppGpp synthetase I encoded by the relA gene [Metzger et al., J. Biol. Chem., 263:15699-15704 (1988); Schreiber et al., J. Biol. Chem., 266:3760-3767 (1991); and Svitil et al., J. Biol. Chem., 268:2307-2311 (1993)]. ppGpp in turn coordinately shuts down the synthesis of macromolecules such as DNA, phospholipids [Sokawa et al., Biochem. Biophys. Res. Commun., 33:108-112 (1968)] and cell wall peptidoglycan [Ishiguro and Ramey, J. Bacteriol., 127:1119-1126 (1976)]. In this setting, antibiotic-induced lysis is blocked by an as yet uncharacterized defect in autolysin activation. This protection from death, termed phenotypic tolerance, is a property of all non-growing bacteria and forms the basis of antibiotic selection for auxotrophs [Hobby et al., Proc. Soc. Exp. Biol., 50:281-285 (1942) and Tuomanen, Revs. Infect. Dis., 8 Suppl. 3:279-291 (1986)]. Phenotypic tolerance during an infection is an important source of residual bacteria that survive antibiotic therapy in vivo, and can thereby promote the subsequent acquisition of antibiotic resistance and the concomitant failure of the antibiotic therapy [Handwerger and Tomasz, Revs. Infect. Dis., 7:368-386 (1985); Novak et al., Nature, 399:590-593 (1999); Tuomanen et al., Antimicrob. Agents Chemother., 30:521-527 (1986); Tuomanen et al., J. Bacteriol, 170:1373-1376 (1988); and Tuomanen et al., J. Infect. Dis., 158:36-43 (1988)].
Antibiotic tolerance, a phenomenon distinct from antibiotic resistance, was first described in 1970 in pneumococci and provided a significant clue to the mechanism of action of penicillin [Tomasz et al., Nature, 227:138-140 (1970)]. Tolerant strains stop growing in the presence of conventional concentrations of antibiotic, but do not subsequently die. Tolerance arises when the bacterial autolytic enzymes, i.e., autolysins, fail to be triggered as the antibiotic inhibits the cell wall synthetic machinery. This explicitly implies that penicillin kills bacteria by activating a set of endogenous hydrolytic enzymes and that bacteria exhibit strategies to stop this activation resulting in survival of antibiotic therapy.
Tolerance is of clinical significance since it has been shown that the inability to eradicate tolerant bacteria leads to failure of antibiotic therapy in clinical infections [Handwerger and Tomasz, Rev. Infect. Dis., 7:368-386 (1985); Tuomanen E., Rev. Infect. Dis., 3:S279-S291 (1986); and Tuomanen et al., J. Infect. Dis., 158:36-43 (1988)]. Furthermore, tolerance is thought to be a prerequisite to the development of antibiotic resistance since it creates survivors of antibiotic therapy. These survivors can then acquire new genetic elements of resistance which allow growth in the presence of antibiotics. Virtually all resistant strains also have been shown to be tolerant [Liu and Tomasz, J. Infect. Dis., 152:365-372 (1985)]. Therefore, the identification of novel antibiotics which can lyse these xe2x80x9cantibiotic-tolerantxe2x80x9d bacteria is necessary.
Mechanistically speaking, tolerance arises in two settings: 1) all bacteria become phenotypically tolerant as growth rate decreases [Tuomanen E., Revs. Infect. Dis., 3:S279-S291 (1986)] and 2) some bacteria are genotypically tolerant by virtue of acquisition of mutations. In both cases, the basic phenomenon is the down regulation of autolysin triggering. This down regulation is transient in phenotypic tolerance in response to environmental cues and is permanent in genotypic tolerance where mutation has changed the lysis control loop. Obviously, the simplest example of genotypic tolerance is the deletion of the autolytic enzymes. This artificial situation was the basis of the first tolerant mutant described in 1970 [Tomasz et al., Nature, 227:138-140 (1970)] but for reasons that remain unclear, no clinical isolates have been found which are tolerant because of deletion of these suicidal enzymes. Rather, clinical tolerance arises at the level of regulation of autolysin activity [Tuomanen et al., J. Infect. Dis., 158:36-43 (1988) and Tuomanen et al., Escherichia coli. J. Bacteriol., 170:1373-1376 (1988)].
The most striking examples of powerful regulation of autolysis occur during bacterial response to stress: the stringent response to nutrient deprivation and the heat shock response. The existence of stress-induced global regulators of autolysis described are indicative of strong negative controls on hydrolase deregulation. Thus, bacteria control autolytic activity in order to prevent suicidal lysis. On the other hand, a striking beneficial clinical effect would accrue if one were able to prevent the generation of this protective response in bacteria, particularly in the case of recalcitrant infections involving bacteria sequestered in areas deficient in growth requirements, such as the cerebrospinal fluid, joint fluid, aqueous humor, cardiac vegetations, abscesses, and bone. It stands to reason that the course of therapy for all such infections is prolonged by the need to eradicate phenotypically tolerant bacteria to avoid the rapid relapse observed when antibiotic therapy is withdrawn and surviving bacteria begin to multiply once again. By identifying new antibiotics which can lyse these antibiotic-tolerant bacteria, it should be possible to subvert the protective effects on bacterial survival of slow growth rate or genotypic mutation to tolerance in vivo, thereby globally improving the outcome of antibiotic therapy.
Bacteria have developed a complex signaling system that enables the cell to respond swiftly to environmental stress. The histidyl-aspartyl (His-Asp) phosphorelay signal transduction system plays a major role in this signal transduction. There are two key participants in the His-Asp phosphorelay signal transduction system: (I) a sensor histidine kinase, which is generally a transmembrane protein; and (2) a response regulator which mediates changes in gene expression and/or cellular locomotion. The sensor histidine kinase contains a periplasmic or extracellular receptor that detects the external signal, and the sensor histidine kinase then mediates the signal into the cell by activating its corresponding response regulator. The activated response regulator then carries the signal intracellularly to effect the cellular response to the external signal. To date, 23-28 open reading frames have been identified in the Escherichia coli genome as encoding putative sensory histidine kinases, whereas 32 open reading frames have been identified as encoding putative response regulators [Mizuno, DNA Research, 4:161-168 (1997)]. The sensory histidine kinase of the His-Asp phosphorelay signal transduction system contains a specific histidine that is autophosphorylated in the presence of ATP. The sensor histidine kinase transfers the phosphoryl group to a specific aspartyl residue of the response regulator. This phosphoryl transfer activates the response regulator and thereby transduces the signal, allowing the cell to rapidly respond to a particular environmental challenge.
Most bacteria also possess transport ATPases that use the energy derived from their enzymatic hydrolysis of ATP to transport compounds into the cell. In E. coli, for example, the transport ATPases are located in the bacterial inner membrane, and they transport compounds from the periplasmic space into the cell. Transport ATPases are members of a large family of transport proteins termed ABC transporters. The name is derived from a highly conserved ATP-binding cassette contained by all of the members. Generally, ABC transporters are specific for a particular type of molecule (e.g., an amino acid, a sugar, an inorganic ion, a peptide or even a protein). [See, Alberts et al., Molecular Biology of the Cell, 3rd edition, Garland Publishing Inc. (New York) Pages 519-522 (1994)]. Heretofore, the relationship between autolysins, His-Asp phosphorelay systems, and ABC transporters has remained obscure.
Bacteria produce peptides and small organic molecules that kill neighboring bacteria. These bacteriocins are of three varieties based on structure: 1) lantibiotics, 2) nonlantibiotics, and 3) others secreted by virtue of a signal peptide (see Cintas et al., J. Bad., 180:1988-1994 (1998)]. Animals, including insects, also naturally produce peptide antibiotics [Bevins et al., Ann. Rev. Biochem., 59:395-414 (1990)]. These antibiotics have been organized in three structural groups: (1) Cysteine-rich xcex2-sheet peptides; (2) xcex1-helical, amphipathic molecules; and (3) proline-rich peptides [Mayasaki et al., Int. J. of Antimicrob. Agents, 9:269-280 (1998)]. However, the use of such antibiotics to combat resistant bacterial strains is only beginning to be exploited.
New approaches to drug development are necessary to combat the ever-increasing number of antibiotic-resistant pathogens. In addition, new antibiotics need to be identified which will act independently of autolysins (such as the pneumococcal autolysin, LytA). Furthermore, there is a need to provide pharmaceutical compositions containing such new antibiotics in order to more effectively treat bacterial infections and inflammations.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The present invention provides methods of identifying novel antibiotic peptides. In addition, the present invention provides the antibiotics themselves. In a particular embodiment, the peptides can act with another antibiotic, such as penicillin, to synergistically kill slow growing or non-growing bacteria. Included in the present invention are antibiotic peptides that can contain unnatural amino acids and/or are branched or cyclic in structure. In a particular embodiment, the peptide is neither hydrophobic nor cationic. The present invention further provides methods of using the antibiotics in the treatment and prevention of bacterial infections and inflammations.
A key aspect of the present invention are antibiotic peptides comprising a portion of the amino acid sequence of MEFMRKEFHNVLSSGQLLADKRPARDYNRK (SEQ ID NO:48) which is encoded by the nucleic acid sequence SEQ ID NO:45, and more particularly by the nucleic acid sequence SEQ ID NO:54. The present invention therefore provides an isolated nucleic acid encoding a peptide comprising the amino acid sequence of SEQ ID NO:2 with a conservative amino acid substitution and/or comprising the amino acid sequence of SEQ ID NO:44 with a conservative amino acid substitution. In a related aspect, the peptide comprises the amino acid sequence of MRKEFHNVLSSDQLLTDKRPARDYN (SEQ ID NO:61) with a conservative amino acid substitution. Preferably, the peptide is capable of inhibiting growth of both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient (e.g., either lacking LytA or containing a defective LytA). More preferably, the peptide can inhibit the growth of a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the nucleic acid encodes a peptide containing no more than 100 amino acids. In a preferred embodiment of this type, the nucleic acid encodes a peptide that contains no more than 75 amino acids. In another embodiment the nucleic acid encodes a peptide that contains no more than 50 amino acids. In still another embodiment, the nucleic acid encodes a peptide that contains 25 to 35 amino acids. In a preferred embodiment, the nucleic acid encodes a peptide comprising the amino acid sequence of MRKEFHNVLSSGQLLADKRPARDYN (SEQ ID NO:2). In a more preferred embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:1. In another embodiment, the nucleic acid encodes a peptide comprising the amino acid sequence of MRKEFHNVLSSDQLLTDKRPARDYN (SEQ ID NO:61).
A related aspect of the present invention provides an isolated nucleic acid encoding a peptide comprising the amino acid sequence of SEQ ID NO:4 with a conservative amino acid substitution. Preferably, the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. More preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the nucleic acid encodes a peptide containing no more than 100 amino acids. In a preferred embodiment of this type, the nucleic acid encodes a peptide that contains no more than 75 amino acids. In another embodiment the nucleic acid encodes a peptide that contains no more than 50 amino acids. In still another embodiment, the nucleic acid encodes a peptide that contains 25 to 35 amino acids. In a preferred embodiment, the nucleic acid encodes a peptide comprising the amino acid sequence of MRKEFHNVLSAGQLLADKRPARDYN (SEQ ID NO:4). In a more preferred embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:3.
A related aspect of the present invention provides an isolated nucleic acid encoding a peptide comprising the amino acid sequence of SEQ ID NO:44 with a conservative amino acid substitution. Another aspect of the present invention provides an isolated nucleic acid encoding a peptide comprising the amino acid sequence of MRKEFHNVLSSDQLLTDKRPARDYNRK (SEQ ID NO:60). Preferably, the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. More preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the nucleic acid encodes a peptide containing no more than 100 amino acids. In a preferred embodiment of this type, the nucleic acid encodes a peptide that contains no more than 75 amino acids. In another embodiment the nucleic acid encodes a peptide that contains no more than 50 amino acids. In still another embodiment, the nucleic acid encodes a peptide that contains 27 to 40 amino acids. In a preferred embodiment, the nucleic acid encodes a peptide comprising the amino acid sequence of MRKEFHNVLSSGQLLADKRPARDYNRK (SEQ ID NO:44). In a more preferred embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:53. In another embodiment, the nucleic acid encodes a peptide comprising the amino acid sequence of SEQ ID NO:60.
In still another embodiment the nucleic acid encodes a peptide having the amino acid sequence of MRKEFHNVLSSGQLLADKRPARDXN (SEQ ID NO:36), (where X is any amino acid residue) with a conservative amino acid substitution. This peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the nucleic acid encodes a peptide containing no more than 100 amino acids. In a preferred embodiment of this type, the nucleic acid encodes a peptide that contains no more than 75 amino acids. In another embodiment the nucleic acid encodes a peptide that contains no more than 50 amino acids. In still another embodiment, the nucleic acid encodes a peptide that contains 25 to 35 amino acids. In a preferred embodiment, the nucleic acid encodes a peptide comprising the amino acid sequence of MRKEFHNVLSSGQLLADKRPARDXN (SEQ ID NO:36).
The present invention also provides a nucleic acid encoding a peptide containing 7 to 100 amino acids that comprises three contiguous amino acids from the amino acid sequence of SEQ ID NO:2, and/or SEQ ID NO: 61 wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the nucleic acid encodes a peptide that contains 12 to 50 amino acids. In another embodiment, the nucleic acid encodes a peptide that contains 17 to 35 amino acids. In a preferred embodiment of this type, the nucleic acid encodes a peptide having 20 to 30 amino acids. In a more preferred embodiment, the nucleic acid encodes a peptide having 25 amino acids.
The present invention further provides a nucleic acid encoding a peptide containing 7 to 100 amino acids that comprises five contiguous amino acids from the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO: 61, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the nucleic acid encodes a peptide that contains 12 to 50 amino acids. In another embodiment, the nucleic acid encodes a peptide that contains 17 to 35 amino acids. In a preferred embodiment of this type, the nucleic acid encodes a peptide having 20 to 30 amino acids. In a more preferred embodiment, the nucleic acid encodes a peptide having 25 amino acids.
The present invention further provides a nucleic acid encoding a peptide containing 7 to 100 amino acids that comprises seven contiguous amino acids from the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO: 61, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the nucleic acid encodes a peptide that contains 12 to 50 amino acids. In another embodiment, the nucleic acid encodes a peptide that contains 17 to 35 amino acids. In a preferred embodiment of this type, the nucleic acid encodes a peptide having 20 to 30 amino acids. In a more preferred embodiment, the nucleic acid encodes a peptide having 25 amino acids.
The present invention further provides a nucleic acid encoding a peptide containing 12 to 100 amino acids that comprises twelve contiguous amino acids from the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO: 61, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the nucleic acid encodes a peptide that contains 16 to 50 amino acids. In another embodiment, the nucleic acid encodes a peptide that contains 20 to 35 amino acids. In a preferred embodiment of this type, the nucleic acid encodes a peptide having 22 to 28 amino acids. In a more preferred embodiment, the nucleic acid encodes a peptide having 25 amino acids.
The present invention also provides a nucleic acid encoding a peptide containing 8 to 100 amino acids, and comprising the amino acid sequence of
DKRPARDY (SEQ ID NO:40)
or the amino acid sequence of DKRPARDY (SEQ ID NO:40), having a conservative amino acid substitution, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the nucleic acid encodes a peptide that contains 12 to 50 amino acids. In another embodiment, the nucleic acid encodes a peptide that contains 17 to 35 amino acids. In a preferred embodiment of this type, the nucleic acid encodes a peptide having 20 to 30 amino acids. In a more preferred embodiment, the nucleic acid encodes a peptide having 25 amino acids.
The present invention also provides a nucleic acid encoding a peptide containing 7 to 100 amino acids and comprising the amino acid sequence of
RKEFHNV (SEQ ID NO:41)
or the amino acid sequence of RKEFHNV (SEQ ID NO:41) having a conservative amino acid substitution, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the nucleic acid encodes a peptide that contains 12 to 50 amino acids. In another embodiment, the nucleic acid encodes a peptide that contains 17 to 35 amino acids. In a preferred embodiment of this type, the nucleic acid encodes a peptide having 20 to 30 amino acids. In a more preferred embodiment, the nucleic acid encodes a peptide having 25 amino acids.
The present invention also provides a nucleic acid encoding a peptide containing 7 to 100 amino acids and comprising the amino acid sequence of
LSSGQLL (SEQ ID NO:42)
or the amino acid sequence of LSSGQLL (SEQ ID NO:42) having a conservative amino acid substitution, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the nucleic acid encodes a peptide that contains 12 to 50 amino acids. In another embodiment, the nucleic acid encodes a peptide that contains 17 to 35 amino acids. In a preferred embodiment of this type, the nucleic acid encodes a peptide having 20 to 30 amino acids. In a more preferred embodiment, the nucleic acid encodes a peptide having 25 amino acids.
The present invention also provides a nucleic acid encoding a peptide containing 23 to 100 amino acids and comprising the amino acid sequence of
RKEFHXXXXXXQLLXDKRPXRDY (SEQ ID NO:39)
(where X can be any amino acid) or this amino acid sequence having a conservative amino acid substitution, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the nucleic acid encodes a peptide that contains no more than 75 amino acids. In another such embodiment the nucleic acid encodes a peptide that contains no more than 50 amino acids. In still another such embodiment, the nucleic acid encodes a peptide that contains 25 to 35 amino acids.
The present invention further provides a nucleic acid encoding a peptide containing 25 to 100 amino acids and comprising an amino acid sequence of
MXXXXXNVLSXGXXXAXXXXAXXXN (SEQ ID NO:43)
or this amino acid sequence having a conservative amino acid substitution, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the nucleic acid encodes a peptide that contains no more than 75 amino acids. In another such embodiment the nucleic acid encodes a peptide that contains no more than 50 amino acids. In still another such embodiment, the nucleic acid encodes a peptide that contains 25 to 35 amino acids.
The present invention further provides nucleic acids encoding the components of the His-Asp phosphorelay pathway and ABC transporter system of the present invention. In one such embodiment, the nucleic acid encodes a histidine kinase having the amino acid sequence of SEQ ID NO:14. In another embodiment the nucleic acid encodes a homologue of that histidine kinase. In still another embodiment the nucleic acid encodes a histidine kinase having the amino acid sequence of SEQ ID NO:14 with a conservative amino acid substitution. In a particular embodiment the nucleic acid has the nucleotide sequence of SEQ ID NO:13. In another embodiment, the nucleic acid encodes a response regulator having the amino acid sequence of SEQ ID NO:16. In another embodiment the nucleic acid encodes a homologue of that response regulator. In yet another embodiment the nucleic acid encodes a response regulator having the amino acid sequence of SEQ ID NO:16 with a conservative amino acid substitution. In a particular embodiment, the nucleic acid has the nucleotide sequence of SEQ ID NO:15.
In a related embodiment, the present invention provides a nucleic acid encoding a component of an ABC transporter system. In one such embodiment the nucleic acid encodes a component having the amino acid sequence of SEQ ID NO:18. In another embodiment, the component is a homologue of that component such as SEQ ID NO:64. In yet another embodiment the component is a homologue of that component such as SEQ ID NO:72. In still another embodiment, the component is a homologue of that component such as SEQ ID NO:74.
In still another embodiment the nucleic acid encodes a component having the amino acid sequence of SEQ ID NO:18 with a conservative amino acid substitution. In yet another embodiment the nucleic acid encodes a component having the amino acid sequence of SEQ ID NO:64 with a conservative amino acid substitution. In still another embodiment the nucleic acid encodes a component having the amino acid sequence of SEQ ID NO:72 with a conservative amino acid substitution. In yet another embodiment the nucleic acid encodes a component having the amino acid sequence of SEQ ID NO:74 with a conservative amino acid substitution.
In a particular embodiment, the nucleic acid has the nucleotide sequence of SEQ ID NO:17. In another embodiment, the nucleic acid encodes a component of the ABC transporter system having the amino acid sequence of SEQ ID NO:20. In another embodiment, the component is a homologue of that component. In yet another embodiment, the nucleic acid encodes a component having the amino acid sequence of SEQ ID NO:20 with a conservative amino acid substitution. In a particular embodiment, the nucleic acid has the nucleotide sequence of SEQ ID NO:19. In another embodiment, the nucleic acid encodes a component of the ABC transporter system having the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:76. In still another embodiment, the component is a homologue of that component. In yet another embodiment, the nucleic acid encodes a component having the amino acid sequence of SEQ ID NO:22 (or SEQ ID NO:76) with a conservative amino acid substitution. In a particular embodiment, the nucleic acid has the nucleotide sequence of SEQ ID NO:21 or SEQ ID NO:75. In another embodiment the nucleic acid has the nucleotide sequence of SEQ ID NO:23. In still another particular embodiment, the nucleic acid has the nucleotide sequence of SEQ ID NO:75. In another embodiment, the nucleic acid has the nucleotide sequence of SEQ ID NO:71. In yet another particular embodiment, the nucleic acid has the nucleotide sequence of SEQ ID NO:73.
All of the nucleic acids of the present invention can also contain an heterologous nucleotide sequence.
The nucleic acids encoding the peptides and proteins of the present invention can be either RNA or DNA. Cloning vectors that comprise such DNAs are therefore also included. Similarly, expression vectors which comprise DNA encoding the peptides or proteins of the present invention, and which are operatively associated with an expression control sequence, are also included. In addition, the present invention contains unicellular hosts that are transfected or transformed with the expression vectors of the present invention. In one such embodiment the unicellular host is a bacterium. The present invention also includes mammalian cells transfected or transformed with the expression vector of the present invention. The present invention further includes method of isolating the peptides and proteins of the present invention prepared by the recombinant methods described herein. Further included in the present invention are the recombinant peptides and proteins isolated by such procedures.
Another aspect of the present invention provides a peptide containing no more than 100 amino acids and comprising the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO: 61 with a conservative amino acid substitution. Preferably, the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. More preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the peptide contains no more than 75 amino acids. In another embodiment the peptide contains no more than 50 amino acids. In still another embodiment, the peptide contains 25 to 35 amino acids. In preferred embodiment, the peptide comprises the amino acid sequence of SEQ ID NO:2. In an alternative embodiment the peptide comprises SEQ ID NO: 61.
A related aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO:4 with a conservative amino acid substitution. Preferably, the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. More preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the peptide contains no more than 100 amino acids. In a preferred embodiment of this type, the peptide contains no more than 75 amino acids. In another embodiment the peptide contains no more than 50 amino acids. In still another embodiment, the peptide contains 25 to 35 amino acids. In a preferred embodiment, the peptide comprises the amino acid sequence of SEQ ID NO:4.
Another related aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO:44. In another embodiment aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO:44 with a conservative amino acid substitution. The present invention also provides a peptide comprising the amino acid sequence of SEQ ID NO:60. In yet another embodiment of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO:60 with a conservative amino acid substitution. Preferably, the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. More preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the peptide contains no more than 100 amino acids. In a preferred embodiment of this type, the peptide contains no more than 75 amino acids. In another embodiment the peptide contains no more than 50 amino acids. In still another embodiment, the peptide contains 27 to 40 amino acids. In a preferred embodiment, the peptide comprises the amino acid sequence of SEQ ID NO:44. In another embodiment, the peptide comprises the amino acid sequence of SEQ ID NO:60.
In another embodiment the present invention provides a peptide having the amino acid sequence of MRKEFHNVLSSGQLLADKRPARDXN (SEQ ID NO:36), (where X is any amino acid residue) with a conservative amino acid substitution. This peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the peptide contains no more than 100 amino acids. In a preferred embodiment of this type, the peptide contains no more than 75 amino acids. In another embodiment the peptide contains no more than 50 amino acids. In still another embodiment, the peptide contains 25 to 35 amino acids. In a preferred embodiment, the peptide comprises the amino acid sequence of MRKEFHNVLSSGQLLADKRPARDXN (SEQ ID NO:36).
The present invention also provides a peptide containing 7 to 100 amino acids that comprises three contiguous amino acids from the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO: 61, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the peptide contains 12 to 50 amino acids. In another embodiment, the peptide contains 17 to 35 amino acids. In a preferred embodiment of this type, the peptide has 20 to 30 amino acids. In a more preferred embodiment, the peptide has 25 amino acids.
The present invention further provides a peptide containing 7 to 100 amino acids that comprises five contiguous amino acids from the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO: 61, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the peptide contains 12 to 50 amino acids. In another embodiment, the peptide contains 17 to 35 amino acids. In a preferred embodiment of this type, the peptide has 20 to 30 amino acids. In a more preferred embodiment, the peptide has 25 amino acids.
The present invention further provides a peptide containing 7 to 100 amino acids that comprises seven contiguous amino acids from the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO: 61, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell.
In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the peptide contains 12 to 50 amino acids. In another embodiment, the peptide contains 17 to 35 amino acids. In a preferred embodiment of this type, the peptide has 20 to 30 amino acids. In a more preferred embodiment, the peptide has 25 amino acids.
The present invention further provides a peptide containing 12 to 100 amino acids that comprises twelve contiguous amino acids from the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO: 61, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the peptide contains 16 to 50 amino acids. In another embodiment, the peptide contains 20 to 35 amino acids. In a preferred embodiment of this type, the peptide has 22 to 28 amino acids. In a more preferred embodiment, the peptide has 25 amino acids.
The present invention also provides a peptide containing 8 to 100 amino acids, and comprising the amino acid sequence of
DKRPARDY (SEQ ID NO:40)
or the amino acid sequence of DKRPARDY (SEQ ID NO:40) having a conservative amino acid substitution, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the peptide contains 12 to 50 amino acids. In another embodiment, the peptide contains 17 to 35 amino acids. In a preferred embodiment of this type, the peptide has 20 to 30 amino acids. In a more preferred embodiment, the peptide has 25 amino acids.
The present invention also provides a peptide containing 7 to 100 amino acids and comprises the amino acid sequence of
RKEFHNV (SEQ ID NO:41)
or the amino acid sequence of RKEFHNV (SEQ ID NO:41) having the conservative amino acid substitution, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin-deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the peptide contains 12 to 50 amino acids. In another embodiment, the peptide contains 17 to 35 amino acids. In a preferred embodiment of this type, the peptide has 20 to 30 amino acids. In a more preferred embodiment, the peptide has 25 amino acids.
The present invention also provides a peptide containing 7 to 100 amino acids and comprises the amino acid sequence of
LSSGQLL (SEQ ID NO:42)
or the amino acid sequence of LSSGQLL (SEQ ID NO:42) having a conservative amino acid substitution, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment the peptide contains 12 to 50 amino acids. In another embodiment, the peptide contains 17 to 35 amino acids. In a preferred embodiment of this type, the peptide has 20 to 30 amino acids. In a more preferred embodiment, the peptide has 25 amino acids.
The present invention also provides a peptide containing 23 to 100 amino acids and comprising the amino acid sequence of
RKEFHXXXXXXQLLXDKRPXRDY (SEQ ID NO:39)
or this amino acid sequence having a conservative amino acid substitution, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the peptide contains no more than 75 amino acids. In another such embodiment the peptide contains no more than 50 amino acids. In still another such embodiment, the peptide that contains 25 to 35 amino acids.
The present invention further provides a peptide containing 25 to 100 amino acids and comprising the amino acid sequence of
MXXXXXNVLSXGXXXAXXXXAXXXN (SEQ ID NO:43)
or this amino acid sequence having a conservative amino acid substitution, wherein the peptide is capable of inhibiting the growth of or killing both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. Preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a particular embodiment of this type, the peptide contains no more than 75 amino acids. In another such embodiment the peptide contains no more than 50 amino acids. In still another such embodiment, the peptide contains 25 to 35 amino acids.
The present invention further provides the components of the His-Asp phosphorelay pathway and ABC transporter system of the present invention. One such embodiment is a histidine kinase having the amino acid sequence of SEQ ID NO:14. Another embodiment is a homologue of that histidine kinase. Still another embodiment is a histidine kinase having the amino acid sequence of SEQ ID NO:14 with a conservative amino acid substitution. Another such embodiment is a response regulator having the amino acid sequence of SEQ ID NO:16. Still another embodiment is a homologue of that response regulator. Yet another embodiment is a response regulator having the amino acid sequence of SEQ ID NO:16 with a conservative amino acid substitution.
In a related embodiment, the present invention provides a component of an ABC transporter system. One such embodiment is a component having the amino acid sequence of SEQ ID NO:18. Another embodiment is a component that is a homologue of the component having the amino acid sequence of SEQ ID NO:18, such as SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:74. Still another embodiment is a component having the amino acid sequence of SEQ ID NO:18 with a conservative amino acid substitution. Yet another embodiment is a component having the amino acid sequence of SEQ ID NO:64 with a conservative amino acid substitution.
Another embodiment is a component of the ABC transporter system having the amino acid sequence of SEQ ID NO:20. Still another embodiment is a homologue of the component having an amino acid sequence of SEQ ID NO:20. Yet another embodiment is a component having the amino acid sequence of SEQ ID NO:20 with a conservative amino acid substitution. Still another embodiment is a component of the ABC transporter system having the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:76. Yet another embodiment is a component that is a homologue of the component having the amino acid sequence of SEQ ID NO:22 and/or SEQ ID NO:76. Yet another embodiment is a component having the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:76 with a conservative amino acid substitution.
All of the proteins of the present invention can also be formed into fusion proteins or chimeric proteins. Fragments (e.g. by proteolytic digestion such as by trypsin) of these proteins are also part of the present invention.
The present invention also provides antibodies raised against any of the proteins or peptides of the present invention. In one such embodiment the antibody is raised against a peptide containing no more than 100 amino acids and comprising the amino acid sequence of SEQ ID NO:2. In another such embodiment the antibody is raised against a peptide containing no more than 100 amino acids and comprises the amino acid sequence SEQ ID NO:2 with a conservative amino acid substitution. In yet another such embodiment the antibody is raised against a peptide containing no more than 100 amino acids and comprises the amino acid sequence SEQ ID NO: 61. In still another such embodiment the antibody is raised against a peptide containing no more than 100 amino acids and comprises the amino acid sequence SEQ ID NO:61 with a conservative amino acid substitution. In a particular embodiment of this type, the antibody is raised against a peptide containing no more than 100 amino acids and comprising the amino acid sequence of SEQ ID NO:44 with a conservative amino acid substitution. In another embodiment of this type, the antibody is raised against a peptide containing no more than 100 amino acids and comprising the amino acid sequence of SEQ ID NO:60 with a conservative amino acid substitution. Preferably the peptide can inhibit the growth of or kill both wild type pneumococci, and a strain of pneumococcus that is autolysin deficient. More preferably, the peptide can inhibit the growth of or kill a vancomycin tolerant bacterial cell. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In a preferred embodiment of this type, the antibody is raised against the peptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:44, SEQ ID NO:60, or SEQ ID NO:61. In a related embodiment, the antibody is raised against a fragment of 6 to 18 contiguous amino acids of the peptide having the amino acid sequence of SEQ ID NO:2. In yet another embodiment, the antibody is raised against the histidine kinase having the amino acid sequence of SEQ ID NO:14, or raised against a fragment thereof.
The antibodies of the present invention can be either polyclonal or monoclonal antibodies, including chimeric antibodies. One embodiment includes an immortal cell line that produces a monoclonal antibody raised against a peptide of the present invention. In a preferred embodiment of this type the monoclonal antibody is raised against a peptide comprising the amino acid sequence of SEQ ID NO:2, or a fragment thereof. In another embodiment, the monoclonal antibody is raised against the response regulator having the amino acid sequence of SEQ ID NO:16, or raised against a fragment thereof.
The present invention further provides pharmaceutical compositions for treating a bacterial infection comprising one or more of the peptides of the present invention, and a pharmaceutically acceptable carrier. Any of the peptides disclosed herein can be used in such pharmaceutical compositions. In one such embodiment, the pharmaceutical composition comprises a peptide having the amino acid sequence of SEQ ID NO:2, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises a peptide having the amino acid sequence of SEQ ID NO:44, and a pharmaceutically acceptable carrier. In yet another embodiment, the pharmaceutical composition comprises a peptide having the amino acid sequence of SEQ ID NO:61, and a pharmaceutically acceptable carrier. In still another embodiment, the pharmaceutical composition comprises a peptide having the amino acid sequence of SEQ ID NO:60, and a pharmaceutically acceptable carrier. In a related embodiment, the pharmaceutical composition can further comprise a second antibiotic such as penicillin, or multiple antibiotics and/or peptides.
The present invention further provides methods of treating or preventing bacterial infections or inflammations comprising administering a pharmaceutical composition of the present invention. Such administration can be performed by any number of means including topically, by injection, or orally.
Still another aspect of the present invention provides methods for identifying peptides that can inhibit the growth of and/or kill a strain of bacteria. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
One such method comprises locating an open reading frame in a gene cluster of a prokaryotic or fungal DNA which encodes two or more components involved in an His-Asp phosphorelay signal transduction system. Preferably the gene cluster is next to another gene cluster encoding one or more components of an ABC transporter system. In another such embodiment, an open reading frame is located in a gene cluster of a prokaryotic or fungal DNA which encodes two or more components of an ABC transporter system. In the Examples below, the DNA is obtained from a bacterial genome. In a particular embodiment the gene cluster encodes at least one of the following: a sensor histidine kinase, or a response regulator. In a preferred embodiment of this type, the histidine kinase is a homologue of the histidine kinase having the amino acid sequence of SEQ ID NO:14. In another embodiment, the response regulator is a homologue of the response regulator having the amino acid sequence of SEQ ID NO:16. In still another embodiment the component of the ABC transporter system is a homologue of the component having the amino acid sequence of SEQ ID NO:18, such as SEQ ID NO:64 or SEQ ID NO:72 or SEQ ID NO:74. In a related embodiment the component of the ABC transporter system is a homologue of the component having the amino acid sequence of SEQ ID NO:20. In another embodiment, the component of the ABC transporter system is a homologue of the component having the amino acid sequence of SEQ ID NO:22 and/or SEQ ID NO:76.
The method can further comprise making the peptide which is encoded by the open reading frame, or a peptide analog thereof, and then testing the peptide for its ability to inhibit the growth of or kill the strain of bacteria. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogues thereof) in a synergistic manner to kill bacterial cells.
In one such embodiment a particular peptide, or analog thereof is identified when it can inhibit the growth of and/or kill a bacterial or fungal cell. In another such embodiment, the peptide can kill bacteria without lysis. In a preferred embodiment, the peptide can act synergistically with penicillin to kill cells.
The peptides of the present invention can be prepared through recombinant means, proteolytic digestions, or preferably chemical synthesis. Analogs of the peptides can, for example, contain portions of the amino acid sequence encoded by the open reading frame alone, or alternatively a portion of the amino acid sequence can be linked together in a fusion peptide/protein. Thus, modification of the peptides of the present invention can also be made in order to make the peptide more stable, or more potent etc. Such modifications may include the use of unnatural amino acids as described below.
The method for identifying peptides that can inhibit the growth of and/or kill a strain of bacteria can further comprise testing the peptide for its ability to inhibit the growth of and/or kill an alternative strain (or species) of a bacterium, including a vancomycin tolerant strain. In a particular embodiment, the peptide is identified when it can inhibit the growth of and/or kill both strains of the bacterium. In one such embodiment, one strain is a wild type strain, and the other strain is a corresponding mutant strain. In a preferred embodiment of this type, the mutant strain lacks an autolysin or contains a defective autolysin. In one such embodiment, the autolysin (e.g., the missing or defective autolysin) is LytA. In a preferred embodiment, the bacterium is a Streptococcus pneumoniae. 
The present invention further provides alternative methods of identifying a peptide that can kill a wild type strain of bacterium. In a particular embodiment the peptide kills autolysis prone pneumococci without lysing the cell. In a preferred embodiment, the peptide acts together with penicillin (or analogs thereof) in a synergistic manner to kill bacterial cells.
One such embodiment comprises locating an open reading frame in a bacterial genome which is within three kilobases of another open reading frame which encodes an ABC transporter system. The peptide encoded by the open reading frame is obtained, (e.g., made by peptide synthesis or through the expression of a recombinant nucleic acid encoding the peptide or alternatively isolated from its natural source), and then tested for its ability to inhibit the growth of and/or kill a wild type strain of a bacterium. The peptide is identified when it can inhibit the growth of and/or kill the bacterium.
In a preferred embodiment of this type the open reading frame encoding the peptide is within one kilobase of the open reading frame which encodes a component of an ABC transporter system. In a more preferred embodiment the open reading frame encoding the peptide is within 500 bases of the open reading frame which encodes a component of an ABC transporter system. In an even more preferred embodiment the peptide is co-transcribed with a component of the ABC transporter system. In a particular embodiment, the component of the ABC transporter system is a homologue of the ABC transporter having the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:64 or SEQ ID NO:72 or SEQ ID NO:74. In another embodiment, the component of the ABC transporter system is a homologue of the ABC transporter having the amino acid sequence of SEQ ID NO:20. In yet another embodiment, the component of the ABC transporter system is a homologue of the ABC transporter having the amino acid sequence of SEQ ID NO:22 and/or SEQ ID NO:76.
In a related embodiment the open reading frame encoding the peptide is also within three kilobases of an open reading frame that encodes a component involved in the His-Asp phosphorelay signal transduction system. In one such embodiment the component involved in the His-Asp phosphorelay signal transduction system is a sensor histidine kinase. In a particular embodiment the sensor histidine kinase is a homologue to the sensor histidine kinase having the amino acid sequence of SEQ ID NO:14. In another such embodiment the component involved in the His-Asp phosphorelay signal transduction system is a response regulator. In a particular embodiment, the response regulator is a homologue of the response regulator having the amino acid sequence of SEQ ID NO:16.
In a preferred embodiment of this type the open reading frame encoding the peptide is within one kilobase of the open reading frame which encodes a component involved in the His-Asp phosphorelay signal transduction system. In a more preferred embodiment the open reading frame encoding the peptide is within 500 bases of the open reading frame which encodes a component involved in the His-Asp phosphorelay signal transduction system. In an alternative embodiment the peptide is co-transcribed with a component of the His-Asp phosphorelay signal transduction system.
In a particular embodiment the method can further comprise testing the peptide for its ability to inhibit the growth of or kill a strain of bacterium that is deficient in an autolysin. In this case the peptide is identified when it can inhibit the growth of or kill both wild type and the autolysin deficient strain of bacterium. Alternatively, the peptide can be tested for its ability kill autolysis prone pneumococci without lysing the cell. In still another embodiment, the peptide is tested for acting synergistically with penicillin (or analogues thereof) for killing bacterial cells. The peptides can be selected for either killing autolysis prone pneumococci without lysing the cell or for acting synergistically with penicillin or an analogue thereof.
In the methods of the present invention for identifying such peptides candidate peptides can be located in the genome of any prokaryotic or fungal cell and preferably a bacterial cell including but not limited to Pneumococcus, Methanococcus, Haemophilus, Archaeoglobus, Borrelia, Synedrocyptis, Mycobacteria, Staphylococcus, and Enterococcus.
The present invention further provides alternative methods of identifying an agent (or drug) that is capable of inhibiting the growth of and/or killing bacterial cells. Alternatively, the peptide can be tested for its ability kill autolysis prone pneumococci without killing the cell.
In still another embodiment, the peptide is tested for acting synergistically with penicillin (or analogues thereof) for killing bacterial cells. One such method includes contacting an agent with a bacterial cell that has a defective His-Asp signaling system and then determining whether the cell stops growing or is killed. An agent (or drug) is identified as being capable of killing a bacterial cell if it kills the bacterial cell or inhibits the growth of the cell. In a preferred embodiment of this type the bacterial cell is a vancomycin tolerant cell. In another preferred embodiment, the defective His-Asp signaling system of the bacterial cell is not inhibited or not killed by a peptide having the amino acid sequence of SEQ ID NO:2. In yet another preferred embodiment, the defective His-Asp signaling system of the bacterial cell is not inhibited or not killed by a peptide having the amino acid sequence of SEQ ID NO:2. In still another, the cell is both tolerant to vancomycin, and in addition is not killed or not inhibited by a peptide having the amino acid sequence of SEQ ID NO:2. In another preferred embodiment, the defective His-Asp signaling system of the bacterial cell is not inhibited or not killed by a peptide having the amino acid sequence of SEQ ID NO:60. In yet another preferred embodiment, the defective His-Asp signaling system of the bacterial cell is not inhibited or not killed by a peptide having the amino acid sequence of SEQ ID NO:60. In still another, the cell is both tolerant to vancomycin, and in addition is not killed or not inhibited by a peptide having the amino acid sequence of SEQ ID NO:60.
As in the methods described above, the cell can be a prokaryotic or fungal cell but is preferably a bacterial cell and is more preferably a pneumococcal cell.
In a particular embodiment the His-Asp signaling system lacks a functional sensor histidine kinase. In a preferred embodiment of this type the sensor histidine kinase has a wild type amino acid sequence of SEQ ID NO:14 or is a homologue thereof. In another embodiment the His-Asp signaling system lacks a functional response regulator. In a preferred embodiment of this type the response regulator has a wild type amino acid sequence of SEQ ID NO:16 or is a homologue thereof. In still another embodiment the His-Asp signaling system lacks both a functional sensor histidine kinase and a functional response regulator.
In a related embodiment the present invention includes a method of identifying an agent that is capable of killing and/or inhibiting the growth of a bacterial cell. Alternatively, the peptide can be tested for its ability to kill autolysis prone pneumococci without lysing the cell. In still another embodiment, the peptide is tested for acting synergistically with penicillin (or analogues thereof) for killing bacterial cells. One such method includes contacting the agent with a bacterial cell that has a defective ABC transporter system and determining whether the cell is inhibited or killed. An agent is identified as being capable of inhibiting the growth of a bacterial cell when the growth of the bacterial cell is inhibited. Similarly, an agent is identified as being capable of killing a bacterial cell when the bacterial cell is killed. In a particular embodiment of this type, the killing of the cell is monitored at about 620 nm (for the optical density of the cell culture) and an agent is identified as being capable of killing a bacterial cell when the optical density at 620 nm of a cell culture is decreased in the presence of an agent. In a preferred embodiment the bacterial cell is a vancomycin tolerant cell. As above, any bacterial cell can be used in this assay but preferably the bacterial cell is a pneumococcal cell.
In one such embodiment the ABC transporter system lacks a functional component that has a wild type amino acid sequence of SEQ ID NO:18 or is a homologue thereof such as SEQ ID NO:64 or SEQ ID NO:72 or SEQ ID NO:74. In another such embodiment the ABC transporter system lacks a functional component that has a wild type amino acid sequence of SEQ ID NO:20 or is a homologue thereof. In still another such embodiment the ABC transporter system lacks a functional component that has a wild type amino acid sequence of SEQ ID NO:22 and/or SEQ ID NO:76 or is a homologue thereof.
The present invention further includes recombinant bacterial cells that lack one or more of the functional components (e.g., a sensor histidine kinase, response regulator and/or a component of the ABC transporter system) described above.
In one such embodiment the cell has been altered so as to have a defective His-Asp phosphorelay system, and the cell is not killed by a peptide having the amino acid sequence of SEQ ID NO:2. Preferably the cell is a bacterial cell. In a particular embodiment the cell is not killed by penicillin. In another embodiment, the cell is a vancomycin tolerant cell. The bacterial cell can be any bacterial cell including but not limited to Pneumococcus, Methanococcus, Haemophilus, Archaeoglobus, Borrelia, and Syndedrocyptis. Preferably the bacterial cell is a pneumococcal cell. In a particular embodiment the His-Asp phosphorelay pathway of the bacterial cell lacks a functional sensor histidine kinase having a wild type amino acid sequence of SEQ ID NO:14. In another such embodiment the His-Asp phosphorelay pathway lacks a functional response regulator having a wild type amino acid sequence of SEQ ID NO:16.
Alternatively the cell has been altered so as to have a defective ABC transporter system, and the cell is not killed by a peptide having the amino acid sequence of SEQ ID NO:2. Preferably the cell is a bacterial cell. In a particular embodiment the cell is not killed by penicillin. In another embodiment the cell is a vancomycin tolerant cell. Again the bacterial cell can be any bacterial cell including but not limited to Pneumococcus, Methanococcus, Haemophilus, Archaeoglobus, Borrelia, and Syndedrocyptis. Preferably the bacterial cell is a pneumococcal cell. In a particular embodiment the ABC transporter system lacks a functional component having a wild type amino acid sequence of SEQ ID NO:18 or SEQ ID NO:64 or SEQ ID NO:72 or SEQ ID NO:74. In another embodiment the ABC transporter system lacks a functional component having a wild type amino acid sequence of SEQ ID NO:20. In still another embodiment the ABC transporter system lacks a functional component having a wild type amino acid sequence of SEQ ID NO:22 and/or SEQ ID NO:76.
The present invention also provides a method of identifying a cell that contains a mutation in a histidine kinase gene. One such embodiment comprises preparing a PCR amplification product for a nucleic acid using a primer for the histidine kinase gene and comparing the PCR amplification product with a control amplification product prepared using the primer and a control nucleic acid encoding the wild type amino acid sequence of the histidine kinase. When the comparing indicates a difference, the cell is identified as containing a mutation in the histidine kinase gene. In a particular embodiment the nucleic acid is obtained from the pneumococcal cell. Preferably the control nucleic acid encodes the amino acid sequence of SEQ ID NO:14. More preferably the control nucleic acid has the nucleotide sequence of SEQ ID NO:13. In one such embodiment the comparing includes the evaluating of the PCR amplification products by single strand conformation polymorphism (SSCP). In another such embodiment the comparing is performed by Restriction Fragment Length Polymorphism (RFLP). In one embodiment the cell is a vancomycin tolerant cell. In a preferred embodiment the cell is a pneumococcal cell.
The present invention also provides a method of identifying a cell that contains a mutation in a response regulator gene. One such embodiment comprises preparing a PCR amplification product for a nucleic acid using a primer for the response regulator gene and comparing the PCR amplification product with a control amplification product prepared using the primer and a control nucleic acid encoding the wild type amino acid sequence of the response regulator. When the comparing indicates a difference, the cell is identified as containing a mutation in the response regulator gene. In a particular embodiment the nucleic acid is obtained from the pneumococcal cell. Preferably the control nucleic acid encodes the amino acid sequence of SEQ ID NO:16. More preferably the control nucleic acid has the nucleotide sequence of SEQ ID NO:15. In one such embodiment the comparing includes the evaluating of the PCR amplification products by single strand conformation polymorphism (SSCP). In another such embodiment the comparing is performed by Restriction Fragment Length Polymorphism (RFLP). In one embodiment the cell is a vancomycin tolerant cell. In a preferred embodiment the cell is a pneumococcal cell.
The present invention also provides a method of identifying a cell that contains a mutation in a component of a gene for the ABC transporter system. One such embodiment comprises preparing a PCR amplification product for a nucleic acid using a primer for the component gene and comparing the PCR amplification product with a control amplification product prepared using the primer and a control nucleic acid encoding the wild type component sequence. When the comparing indicates a difference, the cell is identified as containing a mutation in a gene for a component of the ABC transporter system. In a particular embodiment the nucleic acid is obtained from the pneumococcal cell. Preferably the control nucleic acid encodes the amino acid sequence of SEQ ID NO:18, SEQ ID NO:64, SEQ ID NO:72, or SEQ ID NO:74. More preferably the control nucleic acid has the nucleotide sequence of SEQ ID NO:17. In another embodiment the control nucleic acid has the nucleotide sequence of SEQ ID NO:71 or SEQ ID NO:73.
In another such embodiment, the control nucleic acid encodes the amino acid sequence of SEQ ID NO:64. Alternatively the control nucleic acid encodes the amino acid sequence of SEQ ID NO:20 and more preferably the control nucleic acid has the nucleotide sequence of SEQ ID NO:19. In another embodiment the control nucleic acid encodes the amino acid sequence of SEQ ID NO:22 and/or SEQ ID NO:76 and more preferably the control nucleic acid has the nucleotide sequence of SEQ ID NO:23 or SEQ ID NO:75. In one such embodiment the comparing includes the evaluating of the PCR amplification products by single strand conformation polymorphism (SSCP). In another such embodiment the comparing is performed by Restriction Fragment Length Polymorphism (RFLP). In one embodiment the cell is a vancomycin tolerant cell. In a preferred embodiment the cell is a pneumococcal cell.
In addition, the present invention further includes all of the peptides, agents (or drugs) identified by the methods of the present invention.
Accordingly, it is a principal object of the present invention to provide a novel peptide antibiotic.
It is a further object of the present invention to provide a peptide that acts synergistically with antibiotics that are active against bacterial cell walls.
More particularly it is a further object of the present invention to provide a peptide that acts synergistically with penicillin to kill slow growing or non-growing bacterial cells.
It is a further object of the present invention to provide a method of identifying new peptide antibiotics by inspection of bacterial genomes.
It is a further object of the present invention to provide methods of testing putative peptide antibiotics to identify new agents useful in preventing bacterial proliferation and/or causing bacterial cell death or lysis.
It is a further object of the present invention to provide nucleic acids encoding the peptides of the present invention.
It is a further object of the present invention to provide an antibody specific for a peptide of the present invention.
It is a further object of the present invention to provide a method of producing a peptide of the present invention, including by chemical synthesis, and through recombinant technology.
It is a further object of the present invention to provide a method of designing putative peptide antibiotics through altering the amino acid and/or nucleic acid sequences of a peptide encoded by an open reading frame that is contained in a gene cluster that encodes at least one protein involved in the His-Asp phosphorelay pathway and an ABC transporter.
It is a further object of the present invention to provide methods of detecting and/or identifying penicillin (or related xcex2 lactams) or vancomycin tolerant bacterial strains.
It is a further object of the present invention to provide a method of treating a disease or preventing a condition caused by bacteria through administering a pharmaceutical composition containing a peptide of the present invention.